Measuring Diffusion Coefficients via Two-photon Fluorescence Recovery After Photobleaching

Summary

In this article we will describe the procedure for measuring diffusion coefficients using multi-photon fluorescence recovery after photobleaching. We will begin by aligning the laser along the optical path to the sample and determining the proper experimental parameters, then continue generating and finally fitting fluorescence recovery curves.

Abstract

Multi-fluorescence recovery after photobleaching is a microscopy technique used to measure the diffusion coefficient (or analogous transport parameters) of macromolecules, and can be applied to both in vitro and in vivo biological systems. Multi-fluorescence recovery after photobleaching is performed by photobleaching a region of interest within a fluorescent sample using an intense laser flash, then attenuating the beam and monitoring the fluorescence as still-fluorescent molecules from outside the region of interest diffuse in to replace the photobleached molecules. We will begin our demonstration by aligning the laser beam through the Pockels Cell (laser modulator) and along the optical path through the laser scan box and objective lens to the sample. For simplicity, we will use a sample of aqueous fluorescent dye. We will then determine the proper experimental parameters for our sample including, monitor and bleaching powers, bleach duration, bin widths (for photon counting), and fluorescence recovery time. Next, we will describe the procedure for taking recovery curves, a process that can be largely automated via LabVIEW (National Instruments, Austin, TX) for enhanced throughput. Finally, the diffusion coefficient is determined by fitting the recovery data to the appropriate mathematical model using a least-squares fitting algorithm, readily programmable using software such as MATLAB (The Mathworks, Natick, MA).

Protocol

1. Align the optics. The key equipment for an MP-FRAP experiment include: a mode-locked laser source, Pockels Cell (for beam modulation), pulse generator, dichroic, objective lens, fluorescence emission filter, gated photomultiplier tube, and a data recording system (photon counter and multichannel scaler). 2. Determine safe monitor powers. Attenuate the laser to a low, but reasonable, power for generating fluorescence within the sample. Focus…

Discussion

The power of multi-photon fluorescence recovery after photobleaching lies in its ability to probe thick samples with 3D resolution. Since its development in the 1990’s, MP-FRAP has been used to determine the diffusion coefficient (or analogous transport parameters) in cell bodies, ex vivo thick tissue slices, and in vivo tissue and interstitium. In this article, we presented the equipment necessary to run an MP-FRAP experiment, as well as the proper procedure for aligning the beam path, setting experime…

Materials

Material Name	Type	Company	Catalogue Number	Comment
Ti:sapphire laser		Spectra Physics	Mai Tai
Pockels Cell		Conoptics	350-80
Laser Scanning Microscope		Olympus	Fluoview
Short pass dichroic mirror		Chroma Technologies	670 DCSX-2P
Photomultiplier tube		Hamamatsu	HC125-02
Photon counter		Stanford Research Systems	SR 400
Multi-channel scaler/averager		Stanford Research Systems	SR 430
Pulse Generator		Stanford Research Systems	DG 535
FITC-BSA		Invitrogen	—